Project Summary/Abstract This study aims to develop a tissue engineered meniscus implant with functional attachments, or entheses, in order to produce a clinically viable alternative to cadaver tissue. The entheses anchor the meniscus into the subchondral bone of the tibia, allowing the meniscus to redistribute loads in the knee generated by walking, jumping, etc. Injuries to the meniscus account for over one million surgeries in the United States every year. The only currently viable surgical technique is the transplant of tissue taken from deceased patients. Tissue engineering provides a promising alternative given its potential for patient speci?city and post-implantation, cell-based remodeling. The author?s lab has previously produced tissue engineered meniscus implants, but the viability of these implants requires the formation of functional entheses. Not only are entheses required for meniscus function, they also allow for easier integration with the body by relying on the superior abilities of bone healing versus soft tissue healing. The entheses have a complex tissue structure that has been shown to mediate stress concentrations between the compliant meniscal tissue and the stiff subchondral bone. Mimicking the structure of native meniscal entheses in tissue engineered constructs will produce viable entheses for a functional tissue engineered meniscus implant. Aim 1 is based on the hypothesis that morphological and biomolecular structuring at the micron scale will correlate with micromechanical gradients that correspond to bulk mechanical properties. The proposed work will examine the microstructure of native meniscal entheses using Raman microspectroscopic mapping, confocal ?uorescent microstrain mapping, and bulk tensile testing on the same sample to create a model for the development of tissue engineered constructs. Aim 2 part A is based on the hypothesis that injecting a cell-seeded high density collagen gel into a cell-seeded partially demineralized bone plug will create a triphasic, mechanically tunable, enthesis-like structure. The proposed work will produce mechanically graded enthesis constructs that mimic mechanical and spatial gradients observed in Aim 1. Aim 2 part B is directed at applying this enthesis construct to previously produced meniscal implants to form a fully functional tissue engineered meniscus. Aim 3 will study these implants in a pilot animal study using sheep. This aim will show the clinical feasibility of the implant from Aim 2 and examine the effects of implantation on the tissue engineered meniscus. This study will provide insight into the enthesis structure and produce tissue engineered menisci that can be used as implants in further studies.